Inhibitor Of Uridine Phosphorylase Research Articles

Bioinformatics of uridine/deoxyuridine paths in Trypanosoma evansi revealed targeting uridine phosphorylase and cytidine deaminase

The differences between the host and parasite metabolic paths could be a hot spot for discovery of antiparasitic targets. In this work, the metabolic paths of uridine and deoxyuridine in camels and the blood protozoan Trypanosoma evansi (T. evansi) were investigated by bioinformatics tools. While a set of de novo and salvage enzymes of uridine were found in camels, T. evansi was lacking uridine kinase and the source of UMP comes through salvage of uridine conversion to uracil. Therefore, inhibition of uridine phosphorylase (UPase) or cytidine deaminase (CDa) will affect the downstream UMP, dUMP and thymidylate synthesis by lowering the levels of uracil and uridine inside the parasite, respectively. Given the presence of two UPases in camel, low sequence similarity between camel and T. evansi UPases and CDase, targeting these enzymes might be without deleterious effects on the host cells.

Abstract 2159: In vitro characterization of PTAU and FU interactions in colon cancer cells

Abstract Background: Fluorouracil (FU) remains a main anticancer therapy for colorectal cancer (CRC) and other solid tumors. The mechanism of action of FU is associated with inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. High toxicity and wide-spread chemoresistance, however, have limited the utilization of FU-based therapies. Five intracellular enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP), and uridine phosphorylase (UP) are key determinants of FU sensitivity or resistance. Combination therapies aimed at improving the bioavailability of FU and reducing host toxicity has been evaluated. Recently, our group developed a new uridine phosphorylase inhibitor, 5-phenylthio-acyclouridine (PTAU) that has shown promise in reducing host toxicity and increasing FU efficacy in vivo. The current studies were to characterize interactions of PTAU and FU in vitro and to assess its effects on key metabolic enzymes in human colon cancer cells. Hypothesis: We hypothesized that inhibition of uridine phosphorylase by PTAU would ameliorate the toxic effects of FU and enhance its efficacy by permitting increased doses of FU. Methods: Three colon cancer cell lines with differing p53 status, HCT116-p53wt (wild-type), HCT116-p53null, and HT-29 (p53-mutant); and a normal colonic epithelial cell line, CRL-1790, were used. Doubling times were determined, and IC50 values for FU were derived from kill curves using MTT assay. Cells were treated for 72 hours with IC50 doses of FU, PTAU (100 μM) or uridine (25 μM), alone or in combination, or with the solvent, DMSO. mRNA levels for DPD, TS, TP, and UP were quantitated by qRT-PCR; for intracellular protein expression, cells were fixed and stained with monoclonal antibodies and analyzed by flow cytometry. Results: FU sensitivity correlated with cell doubling times. Basal expression of FU metabolic enzymes differed between CRC and normal cells. mRNAs for DPD and UP were higher in normal relative to cancer cells, but TS mRNA was higher in cancer cells. FU treatment upregulated mRNA expression of DPD, UP, and TS in cancer cells but not in normal cells. Only in cancer cells, DPD induction by FU was potentiated by PTAU. In contrast, treatment with FU decreased protein levels of DPD, TP, and TS in cancer cells. PTAU induced TS protein expression in cancer cells, an effect that was reduced by co-treatment with FU. Conclusions: FU induces mRNA levels of DPD, UP, and TS in cancer cell lines, but protein levels are reduced; the discrepancy between mRNA and protein levels following FU treatment underscores the need for evaluation of colon cancer samples to assess response to FU therapy. This work was supported by a Supplement of the MSM/TU/UABCCC U54 Partnership grant (3U54-CA118948-09S1) from NIH. Citation Format: Esther A. Suswam, Gaurav Kumar, Hyung-Gyoon Kim, Mohamed Osmar, Mahmoud H. el Kouni, Upender Manne. In vitro characterization of PTAU and FU interactions in colon cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2159.

Human uridine phosphorylase-1 inhibitors: a new approach to ameliorate 5-fluorouracil-induced intestinal mucositis.

5-fluorouracil (5-FU) has been broadly used to treat solid tumors for more than 50 years. One of the major side effects of fluoropyrimidines therapy is oral and intestinal mucositis. Human uridine phosphorylase (hUP) inhibitors have been suggested as modulators of 5-FU toxicity. Therefore, the present study aimed to test the ability of hUP blockers in preventing mucositis induced by 5-FU. We induced intestinal mucositis in Wistar rats with 5-FU, and the intestinal damage was evaluated in presence or absence of two hUP1 inhibitors previously characterized. We examined the loss of weight and diarrhea following the treatment, the villus integrity, uridine levels in plasma, and the neutrophil migration by MPO activity. We found that one of the compounds, 6-hydroxy-4-methyl-1H-pyridin-2-one-3-carbonitrile was efficient to promote intestinal mucosa protection and to inhibit the hUP1 enzyme, increasing the uridine levels in the plasma of animals. However, the loss of body weight, diarrhea intensity or neutrophil migration remained unaffected. Our results bring support to the hUP1 inhibitor strategy as a novel possibility of prevention and treatment of mucositis during the 5-FU chemotherapy, based on the approach of uridine accumulation in plasma and tissues.

Design of Novel Potent Inhibitors of Human Uridine Phosphorylase-1: Synthesis, Inhibition Studies, Thermodynamics, and in Vitro Influence on 5-Fluorouracil Cytotoxicity

Uridine (Urd) is a promising biochemical modulator to reduce host toxicity caused by 5-fluorouracil (5-FU) without impairing its antitumor activity. Elevated doses of Urd are required to achieve a protective effect against 5-FU toxicity, but exogenous administration of Urd is not well-tolerated. Selective inhibitors of human uridine phosphorylase (hUP) have been proposed as a strategy to increase Urd levels. We describe synthesis and characterization of a new class of ligands that inhibit hUP type 1 (hUP1). The design of ligands was based on a possible SN1 catalytic mechanism and as mimics of the carbocation in the transition state of hUP1. The kinetic and thermodynamic profiles showed that the ligands here presented are the most potent in vitro hUP1 inhibitors developed to date. In addition, a lead compound improved the antiproliferative effects of 5-FU on colon cancer cells, accompanied by a reduction of in vitro 5-FU cytotoxicity in aggressive SW-620 cancer cells.

Potent combination therapy for human breast tumors with high doses of 5-fluorouracil: remission and lack of host toxicity

The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host toxicity and enhancing its chemotherapeutic efficacy against human breast tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UP, EC 2.4.2.3), the enzyme responsible for uridine catabolism. SCID mice bearing MDA-MB-468 and MCF-7 human breast tumors were injected intraperitoneally with FUra (50, 200 or 300mg/kg) on days 17, 24, and 31 after tumor cell inoculation. PTAU (120mg/kg), uridine (1,320mg/kg), or their combination was administered orally two or 4h after FUra injection. Another four administrations of PTAU plus uridine were given every 8h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38days. Administration of the maximum tolerated dose (50mg/kg) of 5-fluorouracil (FUra) to SCID mice bearing human breast MDA-MB-468 and MCF-7 adenocarcinoma tumor xenografts reduced tumor weight by 59 and 61%, respectively. Administration of 200mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320mg/kg) alone, 2h following the administration of 200mg/kg FUra, did not rescue from FUra host toxicity as all the mice died. Administration of 120mg/kg PTAU resulted in partial rescue from this lethal dose of FUra as 38% of inoculated mice survived and the tumor weights were reduced by approximately 67%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra. All of the mice survived, and MDA-MB-468 and MCF-7 tumor weights were reduced by 97% and total remission, respectively. Doubling the FUra treatment dose to 400mg/kg in the MDA-MB-468 inoculated mice, with the administration of the adjuvant combination treatment of PTAU plus uridine, was unsuccessful in rescuing from FUra toxicity as all the mice died. Lowering the dose of FUra to 300mg/kg, under the same conditions, resulted in 67% mice survival, and the MCF-7 tumor weights were reduced by 100%. Treatment with uridine alone did not protect from FUra toxicity at 200, 300, and 400mg/kg as all of the mice died. At the higher dose of 300 and 400mg/kg FUra, PTAU alone had no rescuing effect. There was no significant difference between MDA-MB-468 and MCF-7 in their response to the different regimens employed in this study in spite of the fact that MDA-MB-468 is estrogen receptor negative while MCF-7 is estrogen receptor positive. The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combination treatment in raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al. in Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the massive doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side effects associated with such doses of uridine. The combination may also allow the escalation of FUra doses for better chemotherapeutic efficacy against human breast carcinoma, with the possibility of avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

Uridine phosphorylase in biomedical, structural, and functional aspects: A review

The activation of xenobiotics often causes malignant tumor cells to resist chemotherapeutic treatment. Uridine phosphorylase is the key enzyme of pyrimidine metabolism and catalyzes the reversible phosphorylation of uridine with the formation of uracil and ribose-1-phosphate. High-selectivity anticancer agents based on uridine phosphorylase inhibitors are promising for treating both oncological and infection diseases. New medicinal preparations can be predicted and rationally developed only on the basis of detailed biomedical, structural, and functional knowledge about the biomacromolecular target enzyme-drug complex.

Exploring new inhibitors of Plasmodium falciparum purine nucleoside phosphorylase

Plasmodium falciparum purine nucleoside phosphorylase ( PfPNP) has a central role in purine salvage and inhibitors of the enzyme have been shown to have antiplasmodial activity. The enzyme preferentially uses inosine as substrate ( K m = 5 μM, k cat/ K m = 7.4 × 10 4 M −1 s −1), but can also use uridine, albeit less efficiently ( K m = 85 μM, k cat/ K m = 306 M −1 s −1). In an effort to identify new PfPNP inhibitors, two series of compounds were prepared. Series 1 was based on known human uridine phosphorylase inhibitors whilst series 2 was uracil equivalents of purine-based PNP transition state inhibitors. These two series of compounds were assayed for inhibition of both PfPNP activity and growth of P. falciparum. The transition state analogues were found to be moderate inhibitors of PfPNP (most potent compound, K i = 6 μM).

Structure-Guided Design of Novel Inhibitors of Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It also plays a role in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. In some cases, an elevated level of this enzyme in solid tumours is thought to contribute to the selective action of these drugs. Nevertheless, the therapeutic value of these fluoropyrimidine antimetabolites is often limited by their toxicity to normal tissue. To address this shortcoming, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been clinically studied for their ability to moderate the cytotoxic side effects of 5-FU and its derivatives, so as to improve the therapeutic index of these agents. We have determined the high resolution structures of human uridine phosphorylase 1 (hUPP1) in complex with natural ligands and known inhibitors. The structures reveal important details underlying the architecture of hUPP1's active site and the proximate surfaces that influence binding of BAU and analogous acyclouridine compounds. This data provides opportunities for designing more potent inhibitors of this enzyme. For instance, the back wall of the substrate binding pocket is conformationally unique relative to earlier elucidated structures of microbial homologues of UPP. These features can be exploited to develop novel inhibitory compounds with improved efficacy against the human enzyme as a step toward the development of better chemotherapeutic regimens that protect normal tissues with relatively lower UPP activity.

Structural basis for the mechanism of inhibition of uridine phosphorylase from Salmonella typhimurium

The three-dimensional structures of three complexes of Salmonella typhimurium uridine phosphorylase with the inhibitor 2,2′-anhydrouridine, the substrate PO4, and with both the inhibitor 2,2′-anhydrouridine and the substrate PO4 (a binary complex) were studied in detail by X-ray diffraction. The structures of the complexes were refined at 2.38, 1.5, and 1.75 A resolution, respectively. Changes in the three-dimensional structure of the subunits in different crystal structures are considered depending on the presence or absence of the inhibitor molecule and (or) the phosphate ion in the active site of the enzyme. The presence of the phosphate ion in the phosphate-binding site was found to substantially change the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. A comparison showed that the highly flexible loop L9 is unstable. The atomic coordinates of the refined structures of the complexes and the corresponding structure factors were deposited in the Protein Data Bank (their PDB ID codes are 3DD0 and 3C74). The experimental data on the spatial reorganization of the active site caused by changes in its functional state from the unligated to the completely inhibited state suggest the structural basis for the mechanism of inhibition of Salmonella typhimurium uridine phosphorylase.

Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy

BackgroundUridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents.ResultsIn this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.ConclusionThe structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

Uridine prevents the glucose deprivation-induced death of immunostimulated astrocytes via the action of uridine phosphorylase

We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS + IFN-γ-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.

Dose dependent inhibition of uridine phosphorylase (UP) by eniluracil (EU): Was the clinical inferiority of the EU/5-fluorouracil (5-FU) phase III trials due to an unrecognized inhibition of 5-FU anabolism?

2058 Background: Eniluracil (EU) is an irreversible inactivator of dihydropyrimidine dehydrogenase (DPD). EU inhibits DPD dependent 5-fluorouracil (5-FU) catabolism, allowing for oral 5-FU administration with essentially complete bioavailability. Although earlier murine studies suggested an increased antitumor effect when EU was administered before 5-FU, clinical studies used co-administered EU and 5-FU (ratio: 10 EU:1 5-FU) (b.i.d.). These phase III trials demonstrated inferiority compared to a regimen of 5-FU/leukovorin, leading to discontinuation of EU development. We hypothesize that the clinical failure of this EU/5-FU schedule may have resulted from competitive inhibition of 5-FU anabolic (ANA) activation by EU. In this study we examined whether EU could competitively inhibit uridine phosphorylase (UP) or orotate phosphoribosyl transferase (OPRT), the primary ANA enzymes of 5-FU. Methods: The cytoplasmic fraction of human embryonic kidney cells (HEK-293) was used as the source of UP and OPRT. Reverse phase HPLC with radioactivity detection was used to quantify [2-14C]-uracil formation from [2-14C]-uridine (UP activity) and to quantify [6-14C]-FUMP formation from [6-14C]-5-FU (OPRT activity). For UP activity, reaction mixtures consisted of increasing concentrations of EU in phosphate buffer containing 150 μM [2-14C]-uridine. For OPRT activity, reaction mixtures consisted of increasing concentrations of EU in phosphate buffer containing 5 μM [6-14C]-FU and 100 μM benzylacyclouridine (UP inhibitor). Reactions were initiated with addition of UP/OPRT enzyme source and allowed to proceed for 30 min at 37°C. Results: EU displayed dose-dependent competitive inhibition of UP activity (IC50 = 0.375 mM). In particular, the EU/5-FU ratio of 2.5:1 produced approximately 50% inhibition of UP activity. In contrast, EU did not inhibit OPRT activity. Conclusions: This study demonstrates that EU competitively inhibits UP, an important enzyme in 5-FU ANA activation, and suggests that changing the dose ratio and/or increasing the interval between EU and 5-FU administration may be useful in optimizing 5-FU antitumor efficacy. [Table: see text]

Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(Phenylthio)acyclouridine, a uridine phosphorylase inhibitor

The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host-toxicity and enhancing its chemotherapeutic efficacy against human colon tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. SCID mice bearing human colon DLD-1 or HCT-15 tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24 and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg) or their combination was administered orally 2 or 4 h after FUra injection. Another four administrations of PTAU+uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. Administration of the maximum tolerated dose (50 mg/kg) of FUra reduced DLD-1 and HCT-15 tumor weights by 48 and 59%, respectively, at day 38 post implantation. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not alleviate FUra host-toxicity as all the mice died. Administration of 120 mg/kg PTAUresulted in partial rescue from this lethal dose of FUra as 63% of mice survived and tumor weights were reduced by approximately 60%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra as 100% of the mice survived and tumor weights were reduced by 81-82%. Delaying the administration of the combination of PTAU plus uridine to 4 h post FUra treatment was less effective in rescuing from FUra toxicity as only 88% of the mice survived and tumor weights were reduced by only 62%. Administration of PTAU alone, under the same conditions, resulted in a 38% survival rate while the tumor weights were reduced by 47%. Treatment with uridine alone did not protect from FUra toxicity at the dose of 200 mg/kg as all mice died. At the higher dose of 300 mg/kg FUra, neither uridine nor PTAU alone, administered 2 h following the treatment with FUra, had any rescuing effect. On the other hand, the use of the PTAU plus uridine combination reduced the tumor weight by 79%, although this reduction in the tumor weight was accompanied by 37% mortality. There was no significant difference between DLD-1 and HCT-15 in their response to the different regimens employed in this study despite the fact that the tumors have different levels of UrdPase. The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host-toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combinationin raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al., Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the large doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side-effects associated with such doses of uridine. This combination may also allow for the escalation of FUra doses for better chemotherapeutic efficacy against human colon carcinoma while avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

5-(Phenylthio)acyclouridine: a powerful enhancer of oral uridine bioavailability: relevance to chemotherapy with 5-fluorouracil and other uridine rescue regimens

The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the pharmacokinetics and bioavailability of oral uridine. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU is fully absorbed after oral administration with 100% oral bioavailability. Uridine (330, 660 or 1320 mg/kg) and/or PTAU (30, 45, 60, 120, 240 or 480 mg/kg) were orally administered to mice. The plasma levels of uridine, its catabolite uracil, and PTAU were measured using HPLC, and pharmacokinetic analysis was performed. Oral PTAU up to 480 mg/kg per day is not toxic to mice. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg has a prolonged plasma half-life of 2-3 h, and peak plasma PTAU concentrations (C(max)) of 41, 51, 74, 126 and 161 microM with AUCs of 70, 99, 122, 173 and 225 micromol h/l, respectively. Coadministration of uridine with PTAU did not have a significant effect on the pharmacokinetic parameters of plasma PTAU at any of the doses tested. Coadministration of PTAU (30, 45, 60 and 120 or 240 mg/kg) with uridine (330, 660 or 1320 mg/kg) elevated the concentration of plasma uridine over that following the same dose of uridine alone, a result of reduced metabolic clearance of uridine as evidenced by decreased plasma exposure (C(max) and AUC) to uracil. Plasma uridine was elevated with the increase of uridine dose at each PTAU dose tested and no plateau was reached. Coadministration of PTAU at 30, 45, 60, 120 and 240 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9-, 9.9-, 11.7- and 12.5-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol h/l) by 5.7-, 6.8-, 8.2-, 6.3-, and 6.9-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.7-, 2.4-, 2.6-, 5.2- and 4.3- fold, and that of 660 mg/kg uridine by 2.3-, 2.7-, 3.3-, 4.6- and 6.7-fold, respectively. The excellent pharmacokinetic properties of PTAU, and its extraordinary effectiveness in improving the oral bioavailability of uridine, could be useful to rescue or protect from host toxicities of 5-fluorouracil and various chemotherapeutic pyrimidine analogues used in the treatment of cancer and AIDS, as well as in the management of medical disorders that are remedied by the administration of uridine including CNS disorders (e.g. Huntington's disease, bipolar disorder), liver diseases, diabetic neuropathy, cardiac damage, various autoimmune diseases, and transplant rejection.

278 Functional status during and after adjuvant therapy for colorectal cancer

Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 57 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45–91%) and generally spared DNA (range: 0.2–11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

Enhancement of the bioavailability of oral uridine by coadministration of 5-(phenylthio)acyclouridine, a uridine phosphorylase inhibitor: implications for uridine rescue regimens in chemotherapy.

The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the oral bioavailability of uridine. PTAU is a new potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU is not toxic to mice and is fully absorbed after oral administration (100% oral bioavailability). PTAU was administered orally to mice alone or with uridine. The plasma levels of PTAU as well as those of uridine and its catabolite uracil were measured using HPLC, and pharmacokinetic analysis was performed. Co-administration of PTAU with uridine elevated the concentration of plasma uridine in a dose-dependent manner over that resulting from the administration of the same dose of uridine alone, and reduced the clearance of uridine as well as the peak plasma concentration (Cmax) and area under the curve (AUC) of plasma uracil. Coadministration of PTAU at 30, 45 and 60 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9- and 9.9-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol x h/l) by 5.7-, 6.8- and 8.2-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45 and 60 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.8-, 2.6- and 2.8-fold, and that of 660 mg/kg uridine by 2.2-, 2.6- and 3.2-fold, respectively. The effectiveness of PTAU in improving the oral bioavailability of uridine could be useful in the rescue or protection from host toxicities of various chemotherapeutic pyrimidine analogues as well as in the management of medical disorders that are remedied by administration of uridine.

Modulation of the pharmacokinetics of endogenous plasma uridine by 5-(phenylthio)acyclouridine, a uridine phosphorylase inhibitor: implications for chemotherapy.

The purpose of this investigation was to evaluate the ability of oral PTAU, 5-(phenylthio)acyclouridine, to increase the concentration of endogenous plasma uridine. PTAU is a new potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU was administered to mice orally and parenterally. The plasma levels of PTAU as well as those of uridine and its catabolite uracil were measured by HPLC, and pharmacokinetic analysis was performed. PTAU was fully adsorbed after oral administration (over 100% oral bioavailability) and no PTAU metabolites were detected. PTAU administered orally had no apparent toxicity at doses up to 120 mg/kg per day for 5 days. Parenteral administration of PTAU at 30, 45 and 60 mg/kg increased the concentration of endogenous plasma uridine (1.8 +/- 0.2 microM) by approximately six-, seven-, and nine-fold, respectively. Plasma uridine concentration remained higher than control values until 8 h after PTAU administration. Similar results were obtained following oral administration of PTAU. The baseline concentrations of endogenous plasma uridine were increased by approximately six-, seven- and ten-fold by oral administration of PTAU at 30, 45 and 60 mg/kg, respectively, and remained higher than the controls until 8 h after PTAU administration. PTAU did not alter the concentration of endogenous plasma uracil. The effectiveness of the PTAU in elevating and sustaining high plasma uridine concentrations may be useful in rescuing or protecting the host from toxicities of various chemotherapeutic pyrimidine analogues as well as in the management of medical disorders that respond to the administration of uridine.

Uridine Phosphorylase Association with Vimentin: INTRACELLULAR DISTRIBUTION AND LOCALIZATION

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.

Effect of 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase, on plasma concentration of uridine released from 2′,3′,5′-tri- O -acetyluridine, a prodrug of uridine: relevance to uridine rescue in chemotherapy

The purpose of this investigation was to study the effects of combining oral 5-(phenylselenenyl)acyclouridine (PSAU) with 2',3',5'-tri-O-acetyluridine (TAU) on the levels of plasma uridine in mice. PSAU is a new lipophilic and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. PSAU has 100% oral bioavailability and is a powerful enhancer of the bioavailability of oral uridine. TAU is a prodrug of uridine and a far superior source of uridine than uridine itself. Oral TAU was administered to mice alone or with PSAU. The plasma levels of uridine and its catabolites as well as PSAU were measured using HPLC and pharmacokinetic analysis was performed. Oral administration of 2000 mg/kg TAU increased plasma uridine by over 250-fold with an area under the curve (AUC) of 754 micromol x h/l. Coadministration of PSAU at 30 and 120 mg/kg with TAU further improved the bioavailability of plasma uridine resulting from the administration of TAU alone by 1.7- and 3.9-fold, respectively, and reduced the Cmax and AUC of plasma uracil. The exceptional effectiveness of PSAU plus TAU in elevating and sustaining a high plasma uridine concentration could be useful in the management of medical disorders that are remedied by administration of uridine, as well as the rescue or protection from host toxicities of various chemotherapeutic pyrimidine analogues.

5-phenylthioacyclouridine: a potent and specific inhibitor of uridine phosphorylase

5-Phenylthioacyclouridine (PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phenylthiouracil) was synthesized as a highly specific and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3). PTAU has inhibition constant ( K is ) values of 248 and 353 nM towards UrdPase from mouse and human livers, respectively. PTAU was neither an inhibitor nor a substrate for thymidine phosphorylase (EC 2.4.2.4), uridine–cytidine kinase (EC 2.7.1.48), thymidine kinase (EC 2.7.1.21), dihydrouracil dehydrogenase (EC 1.3.1.2), orotate phosphoribosyltransferase (EC 2.4.2.10), or orotidine 5′-monophosphate decarboxylase (EC 4.1.2.23), the enzymes that could utilize the substrate (uridine or thymidine) or products (uracil or thymine) of UrdPase. Different isomers of 5-tolylthiouracil also were synthesized and tested as inhibitors of UrdPase. The meta-substituted isomer was 3- to 4-fold more potent as an inhibitor of UrdPase than the para- or ortho-substituted isomers. These data indicate that the hydrophobic pocket in the active site of UrdPase adjacent to the 5-position of the pyrimidine ring can accommodate the meta-substituted 5-phenyluracils better than the other isomers, leading to improved inhibition. Therefore, it is anticipated that the potency of PTAU can be increased further by the addition of certain hydrophobic groups at the meta position of the phenyl ring. PTAU has potential usefulness in the therapy of cancer and AIDS as well as other pathological and physiological disorders that can be remedied by the administration of uridine.